This invention pertains to the design, fabrication, and uses of an electronic system which can actively carry out and control multi-step and multiplex reactions in macroscopic or microscopic formats. In particular, these reactions include molecular biological reactions, such as nucleic acid hybridizations, nucleic acid amplification, sample preparation, antibody/antigen reactions, clinical diagnostics, combinatorial chemistry and selection, drug screening, oligonucleotide and nucleic acid synthesis, peptide synthesis, biopolymer synthesis, and catalytic reactions. A key feature of the present invention is the ability to control the localized concentration of two or more reaction-dependant molecules and their reaction environment in order to greatly enhance the rate and specificity of the molecular biological reaction.
Molecular biology comprises a wide variety of techniques for the analysis of nucleic acids and proteins, many of which form the basis of clinical diagnostic assays. These techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and separation and purification of nucleic acids and proteins (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Many molecular biology techniques involve carrying out numerous operations on a large number of samples. They are often complex and time consuming, and generally require a high degree of accuracy. Many a technique is limited in its application by a lack of sensitivity, specificity, or reproducibility. For example, problems with sensitivity and specificity have so far limited the practical applications of nucleic acid hybridization.
Nucleic acid hybridization analysis generally involves the detection of a very small numbers of specific target nucleic acids (DNA or RNA) with probes among a large amount of non-target nucleic acids. In order to keep high specificity, hybridization is normally carried out under the most stringent conditions, achieved through various combinations of temperature, salts, detergents, solvents, chaotropic agents, and denaturants.
Multiple sample nucleic acid hybridization analysis has been conducted on a variety of filter and solid support formats (see G. A. Beltz et al., in Methods in Enzymology, Vol. 100, Part B, R. Wu, L. Grossmam, K. Moldave, Eds., Academic Press, New York, Chapter 19, pp. 266-308, 1985). One format, the so-called xe2x80x9cdot blotxe2x80x9d hybridization, involves the non-covalent attachment of target DNAs to a filter, which are subsequently hybridized with a radioisotope labeled probe(s). xe2x80x9cDot blotxe2x80x9d hybridization gained wide-spread use, and many versions were developed (see M. L. M. Anderson and B. D. Young, in Nucleic Acid Hybridizationxe2x80x94A Practical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press, Washington D.C., Chapter 4, pp. 73-111, 1985). The xe2x80x9cdot blotxe2x80x9d hybridization has been further developed for multiple analysis of genomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for the detection of overlapping clones and the construction of genomic maps (G. A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15, 1993).
Another format, the so-called xe2x80x9csandwichxe2x80x9d hybridization, involves attaching oligonucleotide probes covalently to a solid support and using them to capture and detect multiple nucleic acid targets. (M. Ranki et al., Gene, 21, pp. 77-85, 1983; A. M. Palva, T. M. Ranki, and H. E. Soderlund, in UK Patent Application GB 2156074A, Oct. 2, 1985; T. M. Ranki and H. E. Soderlund in U.S. Pat. No. 4,563,419, Jan. 7, 1986; A. D. B. Malcolm and J. A. Langdale, in PCT WO 86/03782, Jul. 3, 1986; Y. Stabinsky, in U.S. Pat. No. 4,751,177, Jan. 14, 1988; T. H. Adams et al., in PCT WO 90/01564, Feb. 22, 1990; R. B. Wallace et al. 6 Nucleic Acid Res. 11, p. 3543, 1979; and B. J. Connor et al., 80 Proc. Natl. Acad. Sci. USA pp. 278-282, 1983). Multiplex versions of these formats are called xe2x80x9creverse dot blotsxe2x80x9d.
Using the current nucleic acid hybridization formats and stringency control methods, it remains difficult to detect low copy number (i.e., 1-100,000) nucleic acid targets even with the most sensitive reporter groups (enzyme, fluorophores, radioisotopes, etc.) and associated detection systems (fluorometers, luminometers, photon counters, scintillation counters, etc.).
This difficulty is caused by several underlying problems associated with direct probe hybridization. One problem relates to the stringency control of hybridization reactions. Hybridization reactions are usually carried out under the stringent conditions in order to achieve hybridization specificity. Methods of stringency control involve primarily the optimization of temperature, ionic strength, and denaturants in hybridization and subsequent washing procedures. Unfortunately, the application of these stringency conditions causes a significant decrease in the number of hybridized probe/target complexes for detection.
Another problem relates to the high complexity of DNA in most samples, particularly in human genomic DNA samples. When a sample is composed of an enormous number of sequences which are closely related to the specific target sequence, even the most unique probe sequence has a large number of partial hybridizations with non-target sequences.
A third problem relates to the unfavorable hybridization dynamics between a probe and its specific target. Even under the best conditions, most hybridization reactions are conducted with relatively low concentrations of probes and target molecules. In addition, a probe often has to compete with the complementary strand for the target nucleic acid.
A fourth problem for most present hybridization formats is the high level of non-specific background signal. This is caused by the affinity of DNA probes to almost any material.
These problems, either individually or in combination, lead to a loss of sensitivity and/or specificity for nucleic acid hybridization in the above described formats. This is unfortunate because the detection of low copy number nucleic acid targets is necessary for most nucleic acid-based clinical diagnostic assays.
Because of the difficulty in detecting low copy number nucleic acid targets, the research community relies heavily on the polymerase chain reaction (PCR) for the amplification of target nucleic acid sequences (see M. A. Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990). The enormous number of target nucleic acid sequences produced by the PCR reaction improves the subsequent direct nucleic acid probe techniques, albeit at the cost of a lengthy and cumbersome procedure.
A distinctive exception to the general difficulty in detecting low copy number target nucleic acid with a direct probe is the in-situ hybridization technique. This technique allows low copy number unique nucleic acid sequences to be detected in individual cells. In the in-situ format, target nucleic acid is naturally confined to the area of a cell (xcx9c20-502 xcexcm2) or a nucleus (xcx9c10 xcexcm2) at a relatively high local concentration. Furthermore, the probe/target hybridization signal is confined to a microscopic and morphologically distinct area; this makes it easier to distinguish a positive signal from artificial or non-specific signals than hybridization on a solid support.
Mimicking the in-situ hybridization in some aspects, new techniques are being developed for carrying out multiple sample nucleic acid hybridization analysis on micro-formatted multiplex or matrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). These methods usually attach specific DNA sequences to very small specific areas of a solid support, such as micro-wells of a DNA chip. These hybridization formats are micro-scale versions of the conventional xe2x80x9creverse dot blotxe2x80x9d and xe2x80x9csandwichxe2x80x9d hybridization systems.
The micro-formatted hybridization can be used to carry out xe2x80x9csequencing by hybridizationxe2x80x9d (SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of all possible n-nucleotide oligomers (n-mers) to identify n-mers in an unknown DNA sample, which are subsequently aligned by algorithm analysis to produce the DNA sequence (R. Drmanac and R. Crkvenjakov, Yugoslav Patent Application #570/87, 1987; R. Drmanac et al., 4 Genomics, 1-14, 1989; Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1991; and R. Drmanac and R. B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13, 1993), U.S. Pat. No. 6,018,041, Jan. 25, 2000, and U.S. Pat. No. 6,025,136, Feb. 15, 2000).
There are two formats for carrying out SBH. One format involves creating an array of all possible n-mers on a support, which is then hybridized with the target sequence. This is a version of the reverse dot blot. Another format involves attaching the target sequence to a support, which is sequentially probed with all possible n-mers. Both formats have the fundamental problems of direct probe hybridizations and additional difficulties related to multiplex hybridizations. This inability to achieve xe2x80x9csequencing by hybridizationxe2x80x9d by a direct hybridization method lead to a so-called xe2x80x9cformat 3xe2x80x9d, which incorporates a ligase reaction step. While, providing some degree of improvement, it actually represents a different mechanism involving an enzyme reaction step to identify base differences.
Southern, United Kingdom Patent Application GB 8810400, 1988; E. M. Southern et al., 13 Genomics 1008, 1992, proposed using the xe2x80x9creverse dot blotxe2x80x9d format to analyze or sequence DNA. Southern identified a known single point mutation using PCR amplified genomic DNA. Southern also described a method for synthesizing an array of oligonucleotides on a solid support for SBH. However, Southern did not address how to achieve optimal stringency condition for each oligonucleotide on an array. See also U.S. Pat. No. 6,054,270, Apr. 25, 2000.
Fodor et al., 364 Nature, pp. 555-556, 1993, used an array of 1,024 8-mer oligonucleotides on a solid support to sequence DNA. In this case, the target DNA was a fluorescently labeled single-stranded 12-mer oligonucleotide containing only nucleotides the A and C bases. A concentration of 1 xcexcmol (xcx9c6xc3x971011 molecules) of the 12-mer target sequence was necessary for the hybridization with the 8-mer oligomers on the array. The results showed many mismatches. Like Southern, Fodor et al., did not address the underlying problems of direct probe hybridization, such as stringency control for multiplex hybridizations. These problems, together with the requirement of a large quantity of the simple 12-mer target, indicate severe limitations to this SBH format.
Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, used the above discussed second format to sequence several short (116 bp) DNA sequences. Target DNAs were attached to membrane supports (xe2x80x9cdot blotxe2x80x9d format). Each filter was sequentially hybridized with 272 labeled 10-mer and 11-mer oligonucleotides. A wide range of stringency conditions were used to achieve specific hybridization for each n-mer probe; washing times varied from 5 minutes to overnight, and temperatures from 0xc2x0 C. to 16xc2x0 C. Most probes required 3 hours of washing at 16xc2x0 C. The filters had to be exposed for 2 to 18 hours in order to detect hybridization signals. The overall false positive hybridization rate was 5% in spite of the simple target sequences, the reduced set of oligomer probes, and the use of the most stringent conditions available.
Fodor et al., 251 Science 767-773, 1991, used photolithographic techniques to synthesize oligonucleotides on a matrix. Pirrung et al., in U.S. Pat. No. 5,143,854, Sep. 1, 1992, teach large scale photolithographic solid phase synthesis of polypeptides in an array fashion on silicon substrates.
In another approach of matrix hybridization, Beattie et al., in The 1992 San Diego Conference: Genetic Recognition, November, 1992, used a microrobotic system to deposit micro-droplets containing specific DNA sequences into individual microfabricated sample wells on a glass substrate. The hybridization in each sample well is detected by interrogating miniature electrode test fixtures, which surround each individual microwell with an alternating current (AC) electric field.
Regardless of the format, all current micro-scale DNA hybridizations and SBH approaches do not overcome the underlying problems associated with nucleic acid hybridization reactions. They require very high levels of relatively short single-stranded target sequences or PCR amplified DNA, and produce a high level of false positive hybridization signals even under the most stringent conditions. In the case of multiplex formats using arrays of short oligonucleotide sequences, it is not possible to optimize the stringency condition for each individual sequence with any conventional approach because the arrays or devices used for these formats can not change or adjust the temperature, ionic strength, or denaturants at an individual location, relative to other locations. Therefore, a common stringency condition must be used for all the sequences on the device. This results in a large number of non-specific and partial hybridizations and severely limits the application of the device. The problem becomes more compounded as the number of different sequences on the array increases, and as the length of the sequences decreases below 10-mers or increase above 20-mers. This is particularly troublesome for SBH, which requires a large number of short oligonucleotide probes.
Nucleic acids of different size, charge, or conformation are routinely separated by electrophoresis techniques which can distinguish hybridization species by their differential mobility in an electric field. Pulse field electrophoresis uses an arrangement of multiple electrodes around a medium (e.g., a gel) to separate very large DNA fragments which cannot be resolved by conventional gel electrophoresis systems (see R. Anand and E. M. Southern in Gel Electrophoresis of Nucleic Acidsxe2x80x94A Practical Approach, 2 ed., D. Rickwood and B. D. Hames Eds., IRL Press, New York, pp. 101-122, 1990).
Pace, U.S. Pat. No. 4,908,112, Mar. 13, 1990, describes using micro-fabrication techniques to produce a capillary gel electrophoresis system on a silicon substrate. Multiple electrodes are incorporated into the system to move molecules through the separation medium within the device.
Soane and Soane, U.S. Pat. No. 5,126,022, Jun. 30, 1992, describe that a number of electrodes can be used to control the linear movement of charged molecules in a mixture through a gel separation medium contained in a tube. Electrodes have to be installed within the tube to control the movement and position of molecules in the separation medium.
Washizu, M. and Kurosawa, O., 26 IEEE Transactions on Industry Applications 6, pp. 1165-1172, 1990, used high-frequency alternating current (AC) fields to orient DNA molecules in electric field lines produced between microfabricated electrodes. However, the use of direct current (DC) fields is prohibitive for their work. Washizu 25 Journal of Electrostatics 109-123, 1990, describes the manipulation of cells and biological molecules using dielectrophoresis. Cells can be fused and biological molecules can be oriented along the electric fields lines produced by AC voltages between the micro-electrode structures. However, the dielectrophoresis process requires a very high frequency AC (1 MHz) voltage and a low conductivity medium. While these techniques can orient DNA molecules of different sizes along the AC field lines, they cannot distinguish between hybridization complexes of the same size.
MacConnell, U.S. Pat. No. 4,787,936, Nov. 29, 1988, describes methods and means for annealing complementary nucleic acid molecules at an accelerated rate. The nucleic acid probes are electrophoretically concentrated with a surface to which various sequences are bound. Unannealed probe molecules are electronically removed from the surface region by reversal of the electrical orientation, so as to electrophoretically move away from the surface of those materials which had been previously concentrated at the surface. In yet another aspect, the patent describes moving concentrated, unannealed probe molecules successively in various directions along the surface to which the sequences are bound.
Stanley, C. J., U.S. Pat. No. 5,527,670, issued Jun. 18, 1996, claiming priority to GB 9019946, filed Sep. 12, 1990 and GB 9112911 filed Jun. 14, 1991. Stanley discloses a process for denaturing native double-stranded nucleic acid material into its individual strands in an electrochemical cell. An electrical treatment of the nucleic acid with a voltage applied to the nucleic acid material by an electrode is utilized. Promoter compounds, such as methylviologen, are suggested to speed denaturation. The process is suggested for use in the detection of nucleic acid by hybridizing with a labeled probe or in the amplification of DNA by a polymerase chain reaction or ligase chain reaction.
More recently, attempts have been made at microchip based nucleic acid arrays to permit the rapid analysis of genetic information by hybridization. Many of these devices take advantage of the sophisticated silicon manufacturing processes developed by the semiconductor industry over the last forty years. In these devices, many parallel hybridizations may occur simultaneously on immobilized capture probes. Stringency and rate of hybridization is generally controlled by temperature and salt concentration of the solutions and washes. Even though of very high probe densities, such a xe2x80x9cpassivexe2x80x9d micro-hybridization approaches have several limitations, particularly for arrays directed at reverse dot blot formats, for base mismatch analysis, and for re-sequencing and sequencing by hybridization applications.
First, as all nucleic acid probes are exposed to the same conditions simultaneously, capture probes must have similar melting temperatures to achieve similar levels of hybrid stringency. This places limitations on the length, GC content and secondary structure of the capture probes. Also, single-stranded target fragments must be selected out for the actual hybridization, and extremely long hybridization and stringency times are required(see, e.g., Guo, Z, et.al., Nucleic Acid Research, V.22, #24, pp 5456-5465, 1994).
Second, for single base mismatch analysis and re-sequencing applications a relatively large number of capture probes ( greater than 16) must be present on the array to interrogate each position in a given target sequence. For example, a 400 base pair target sequence would require an array with over 12,000 different probe sequences (see, e.g., Kozal, M. J., et.al., Nature Medicine, V.2, #7, pp.753-759, 1996).
Third, for many applications large target fragments, including PCR or other amplicons, can not be directly hybridized to the array. Frequently, complicated secondary processing of the amplicons is required, including: (1) further amplification; (2)conversion to single-stranded RNA fragments; (3) size reduction to short oligomers, and (4) intricate molecular biological/enzymatic reactions steps, such as ligation reactions.
Fourth, for passive hybridization the rate is proportional to the initial concentration of the target fragments in the solution, therefore, very high concentrations of target is required to achieve rapid hybridization.
Fifth, because of difficulties controlling hybridization conditions, single base discrimination is generally restricted to capture oligomers sequences of 20 bases or less with centrally placed differences (see, e.g., Chee ""96; Guo, Z, et.al., Nucleic Acid Research, V.22, #24, pp 5456-5465, 1994; Kozal, M. J., et.al., Nature Medicine, V.2, #7, pp.753-759, 1996).
As is apparent from the preceding discussion, numerous attempts have been made to provide effective techniques to conduct multi-step, multiplex hybridizations and other molecular biological reactions. However, for at least the reasons stated above, these techniques have been proved deficient. Despite the long-recognized need for effective technique, no satisfactory solution has been proposed previously.
In an attempt to circumvent these limitations, the present invention utilizes electric fields as an independent parameter to modulate or control multi-step and multiplex reactions in macroscopic or microscopic formats. In particular, these reactions include molecular biological reactions, such as nucleic acid hybridizations, nucleic acid amplification, sample preparation, antibody/antigen reactions, clinical diagnostics, combinatorial chemistry and selection, drug screening, oligonucleotide and nucleic acid synthesis, peptide synthesis, biopolymer synthesis, and catalytic reactions. The devices of the invention have proven particularly useful for the acceleration of transport and hybridization of nucleic acids and the control of stringency of nucleic acid interactions. These are xe2x80x9cactivexe2x80x9d devices in that they exploit electronic instead of passive diffusion technology. The xe2x80x9cactivexe2x80x9d device provides a controllable electric (electrophoretic) field as a driving force to move and concentrate nucleic acid molecules (probes and/or targets) or other reagents to a selected microscopic/macroscopic test site (with other fixed target or probe molecules). In addition to salt, pH, temperature and chaotropic agents, the electric field strength (voltage/current/current density) provides a precisely controllable and continuously variable parameter for adjustment of nucleic acid interactions. Finally, by the utilization of particular buffer compositions on either side of the test site/semi-permeable matrix structure, the devices and methods of the present invention create favorable reaction zones for the reactant molecules (e.g. DNA probes and targets), and the ability to strictly control or modulate the reaction at the test site. Thus, a key aspect of the electronic devices and methods of this invention is that reactants or analytes from a substantially non-reactive environment can be rapidly brought to a selected test site (microlocation/macrolocation) where other selective binding agents or reactants are present and a third group of entities from another physically separate environment can be rapidly brought to the test site to create a favorable reaction zone.
The present invention relates to the design, fabrication, and uses of electronic systems and devices which can actively carry out controlled multi-step and multiplex reactions in microscale and macroscale formats. These reactions include, but are not limited to, most molecular biological procedures, such as nucleic acid hybridizations, antibody/antigen reaction, cell separation, and related clinical diagnostics.
In addition, the devices are able to carry out multi-step combinational biopolymer and combinatorial synthesis, including, but not limited to, the synthesis of different oligonucleotides or peptides at specific microlocations.
In addition, the electronic devices and methods of this invention allow rapid multiplex hybridization and discrimination of single base mismatches in full length DNA fragments and PCR amplicons, under what would be considered substantially non-hybridizing, denaturing or non-stringent conditions by any passive or conventional hybridization technique so that hybridization reactions occur only at the test sites.
In the reaction-modifying methods of the invention, each test site (microlocation, macrolocation) comprises a semi-permeable matrix (SPM) between two buffer reservoirs or chambers. This semi-permeable matrix may comprise several xe2x80x9csub-layersxe2x80x9d with different functions (e.g., a charged surface membrane layer and a hydrogel layer). However, the overall characteristic of the SPM is that it forms a barrier which impedes the free diffusion of molecules between the two buffer reservoirs (chambers). Each of the buffer reservoirs (chambers) has an associated electrode(s). Attached to the surface of or within the SPM is a specific binding entity. In preferred embodiments of the invention, a first buffer reservoir contains a low conductance buffer and a first charged entity which reacts with the specific binding entity. A second buffer reservoir contains a second, oppositely charged entity which affects the reaction between the first charged entity and the specific binding entity. When an electric potential is applied across the SPM, the first charged entity rapidly migrates through bulk solution to the test site/semi-permeable matrix, creating a localized area of high concentration where it interacts with the specific binding entity. At the same time, the second charged entity migrates xe2x80x9cthroughxe2x80x9d the SPM, creating a localized zone of relatively high concentration of the second entity within and adjacent to the SPM wherein it may interact with the first charged entity and the specific binding entity at an effective concentration. Thus, a xe2x80x9creaction zonexe2x80x9d of high localized concentration of the first and second entity (by electromotive force) and the specific binding entity (by binding to the SPM) is created at the semi-permeable interface between the two buffer reservoirs. This greatly facilitates the interaction between the first charged entity and the specific binding entity by first creating a zone of high concentration which increases the number of collisions between the specific binding entity and the two charged entities, and second by producing favorable conditions for the reaction (pH, specific buffer species, specific cations, specific anions, ionic strength, surfactants, etc.).
If the binding reaction is being utilized as a detection method, as in the case of a DNA hybridization reaction, or antigen/antibody interactions, then the method may also comprise a step utilizing the electric field to remove the members of the first charged entity which are reacting with the specific binding entity in a non-specific manner. By reversing the potential/bias, the first charged entity interacting with the specific binding entity experiences an electromotive force away from the specific binding entity. In addition, the concentration of the second charged entity, which had facilitated interactions between the first charged entity and the specific binding entity, decreases as it ceases to migrate through the SPM. Thus, members of the first charged entity which are not bound specifically to the specific binding entity, including single base mismatches which are slightly less thermodynamically stable, will dissociate from the specific binding entity, leaving the specifically bound members of the first charged entity for detection.
The macroscale and microscale devices for use in the methods of the present invention are preferably fabricated using techniques which include micromachining, high-tolerance molding, microlithography or any other techniques used for fabricating small laboratory instruments or systems, lab on a chip devices, or biochip devices. The device of the invention has a test site location comprising a semi-permeable matrix between first and second buffer reservoirs or chambers (FIG. 1). Although a single test location device is considered within the embodiments of the present invention, devices with a multiple of test sites or locations (microlocations or macrolocations) are the more preferred embodiments of this invention (FIG. 2). While the devices of this invention may contain a specific binding entity on or within the SPM, other devices may simply have binding or reactive sites on or within the SPM for later attachment of the specific binding entities, e.g., streptavidin. For instance, in a preferred embodiment, the device for use in the present invention comprises a SPM which contains streptavidin, so that specific binding entities which have been modified with a biotin moiety (e.g. DNA probes or target sequences) may be subsequently addressed and attached to the SPM.
Although a common first and second buffer reservoir (chamber) may be used for all microlocations, individual reservoirs for each location may also be used for the first, second, or both buffer reservoirs (FIGS. 3, 4, and 5). The use of individual first reservoirs (chambers) may, for example, be desirable to prevent cross-contamination of patient samples in high-throughput clinical testing applications of the invention. Alternatively, indentations in a first buffer reservoir (or the support structure for the SPM) may be provided to aid sample loading, as depicted in FIGS. 8 and 9. These applications could still utilize a common second reservoir, as depicted in FIG. 3. Although each individual test site microlocation may be able to electronically control and direct the transport and attachment of specific binding entities (e.g., nucleic acids, antibodies), a device in which common electrodes are used for all test sites microlocations is one of the more preferred embodiments of the present invention (FIG. 2). The use of a first and second common electrode in a first and second common buffer reservoir allow several substantially similar reactions to be carried out in parallel on several individual test sites microlocations under identical electronic conditions. This embodiment is preferable in instances where high-throughput processing of multiple samples is required, such as in pharmaco-genomic, drug discovery and some high volume clinical testing applications. Conversely, individual first and seconds electrodes may be used, as depicted in FIG. 3 or FIG. 4. These embodiments may be preferred in order to individually control the electric field at each test site microlocation, e.g., for electronic stringency in particular. All test sites microlocations can be addressed with different or similar specific binding entities. Because of the use of xe2x80x9cactivexe2x80x9d electronic control over the molecular biochemical reaction and its environment, different specific binding entities and first and second charged entity may be used under conditions (i.e. ambient temperature, pH, ionic strength) where they (the reactants) would not normally be compatible or controllable in a conventional or passive type reactions.
Thus, one aspect of the present invention is a device for carrying out molecular biological reactions with an array of test sites or microlocations (or macrolocations) across which an electric field (current) may be applied, wherein:
a) each microscopic location comprises a SPM separating a first and second buffer reservoir (chamber) containing a first and second charged entity;
b) the SPM is so composed such that there is little or no migration of the first charged entity in the absence of an electric field, but so that controlled migration of the first charged entity occurs when an electric field is applied for the time necessary to achieve the biochemical reaction;
c) the SPM is so composed that there is little or no migration of the second charged entity in the absence of an electric field, but so that controlled migration of the second charged entity occurs when an electric field is applied for the time necessary to achieve the biochemical reaction;
d) wherein at each test site or microlocation there is attached to the first buffer chamber side of the SPM a specific binding entity which reacts with the first charged entity; and
e) wherein the second charged entity modulates the reaction of the first charged entity with the specific binding entity.
By xe2x80x9carrayxe2x80x9d is meant an arrangement of test site microlocations (and/or macrolocations) on the device. The locations can be arranged in two dimensional arrays, three dimensional arrays, or other formats. The number of locations can range from several to hundreds of thousands. In some cases (e.g., high throughput systems) microlocations can be DNA or other samples spotted (addressed) onto filter paper or membranes which are processed through an electronic hybridization system.
In a second aspect, this invention features a method for attaching the specific binding entity to the first-buffer side of the microlocations on the device. When activated, the device can affect the free field electrophoretic transport of any charged functionalized specific binding entity directly to the microlocation. Upon contacting the specific test site microlocation, the functionalized specific binding entity immediately becomes attached (covalently or non-covalently) to the surface or within the SPM. The process can be rapidly carried out in parallel at each test site microlocation, whether common or individual first buffer reservoirs are used.
By xe2x80x9ccharged functionalized specific binding entityxe2x80x9d is meant a specific binding entity that is reactive (i.e., capable of covalent or non-covalent attachment to a location) and carries a net charge (either positive or negative). This also includes mixtures of more than one specific binding entity.
In a third aspect, this invention features a method for concentrating and reacting analytes, probes or reactants within a reaction zone at the test site microlocations (macrolocations) on the device (FIG. 1b). After the attachment of the specific binding entities, the analyte(s), probe(s) or reactant(s) (the first charged entity) is loaded into the first buffer reservoir. A second buffer, containing the second charged entity, is loaded into the second buffer reservoir. An electric potential is applied across the SPM between the two reservoirs, producing subsequent electrophoretic ionic flow across the SPM. This unique feature allows relatively dilute charged analytes or reactant molecules free in solution (first reservoir) to be rapidly transported, concentrated, and reacted at the microlocations while the second charged entity (second reservoir) migrates across the SPM to modulate the reaction between the analyte, probe, or reagent and the specific binding entity. This ability to concentrate dilute analyte or reactant molecules at selected microlocations wherein a reaction zone of increased concentration of the second charged entity is created that greatly accelerates the reaction at these test site microlocations. Thus, in the third aspect of the invention electronic devices and methods relate to improving reaction rates and efficiency. In the case of nucleic acid hybridization reactions, xe2x80x9celectronic hybridizationxe2x80x9d can be carried out according to the above method, the methods further comprising steps chosen from:
using xe2x80x9celectronic hybridizationxe2x80x9d to improve the overall hybridization of amplified target DNA and RNA sequences on arrays of capture probe oligonucleotides.
using xe2x80x9celectronic hybridizationxe2x80x9d to improve the hybridization of any target DNA or RNA sequences on arrays of capture probe oligonucleotides in reverse dot blot formats.
using xe2x80x9celectronic hybridizationxe2x80x9d for sequencing by hybridization (SBH) utilizing arrays with capture probes 8 bases or less in length.
using xe2x80x9celectronic hybridizationxe2x80x9d to improve the hybridization of any target DNA or RNA sequences on arrays of capture probes in sandwich formats, with subsequent hybridization of reporter probes (fluorescent, chemiluminescent, radioactive, etc.).
using xe2x80x9celectronic hybridizationxe2x80x9d to improve the hybridization of any DNA or RNA sequence on arrays of nucleic acid sequences in dot blot formats (target sequences addressed to the array, with subsequent hybridization of reporter probes)
using xe2x80x9celectronic hybridizationxe2x80x9d to improve the hybridization of target nucleic acid sequences on arrays of nucleic acid probes in homogeneous/heterogeneous hybridization formats.
using xe2x80x9celectronic hybridizationxe2x80x9d to improve the hybridization of target RNA or cDNA sequences on arrays of nucleic acid probes for gene expression applications.
using xe2x80x9celectronic hybridizationxe2x80x9d for individual hybridization events occurring in the same bulk solution and at the same temperature.
using xe2x80x9celectronic hybridizationxe2x80x9d to improve hybridization and detection of un-amplified or low copy number target DNA sequences by complexity reduction.
Using xe2x80x9celectronic hybridizationxe2x80x9d to improve signal amplification techniques (fluorescent, chemiluminescent, calorimetric, enzymatic, dendrimers, branched DNA, and metallic, fluorescent, or magnetic nanospheres, etc.) used to detect un-amplified target or poorly amplified target DNA sequences.
When the desired reaction is complete, the electric field potential/bias can be reversed to remove non-specific analytes or unreacted molecules from the microlocations. The subsequent analysis of the analytes at the specific microlocations is also greatly improved by the ability to repulse non-specific entities and partially hybridized sequences from these locations.
Thus, in a fourth aspect, this invention features a methods for improving efficiency and stringency of nucleic acid hybridization reactions as carried out according to the above method, the methods further comprising steps chosen from:
rapidly removing non-specifically bound target DNA (or DNA probe) sequences from specific test site microlocation(s) or macrolocation(s) where hybridization has occurred by reversing the electric potential;
rapidly removing competing complementary target DNA or DNA probe sequences from specific test site microlocation(s) or macrolocation(s) where hybridization has occurred by reversing the electric potential;
adjusting electronic stringency control (ESC) via voltage and current level and density to remove partially hybridized DNA sequences or DNA probe (more than one base mis-match);
adjusting ESC via voltage and current level and density to improve the resolution of single base mis-match hybridizations using probes in the 8-mer to 21-mer range (e.g., to identify point mutations);
using ESC via voltage and current level and density, to utilize oligonucleotide point mutation probes outside of the ranges used in conventional procedures (e.g., probes longer than 21-mers and shorter than 8-mers); for example, 4-mer to 7-mer, and 22-mer to 30-mer or longer.
using ESC via voltage and current level and density, for sequencing by hybridization (SBH) arrays utilizing capture probes from 4 to 8 nucleotides in length.
applying ESC via voltage and current level and density, to discriminate single nucleotide polymorphisms (SNPs).
using ESC to improve the overall hybridization of amplified target DNA and RNA sequences on arrays of capture probe oligonucleotides.
using ESC to improve the hybridization of any target DNA or RNA sequences on arrays of capture probe oligonucleotides in reverse dot blot formats.
using ESC to improve the hybridization of any target DNA or RNA sequences on arrays of capture probe oligonucleotides in sandwich formats.
using ESC to improve the hybridization of any DNA or RNA sequence on arrays of nucleic acid sequences in the more classical dot blot format (target sequences on the array, reporter probes added)
using ESC to improve the hybridization of target nucleic acid sequences on arrays of nucleic acid probes in homogeneous/heterogeneous hybridization formats.
using ESC to improve the hybridization of target RNA or cDNA sequences on arrays of nucleic acid probes for gene expression applications.
applying independent ESC to individual hybridization events occurring in the same bulk solution and at the same temperature; and
using ESC to improve hybridization and detection of un-amplified target DNA or poorly amplified sequences on arrays.
using ESC to improve signal amplification techniques (fluorescent, chemiluminescent, colorimetric, enzymatic, dendrimers, branched DNA, etc.) used to detect un-amplified target or poorly amplified target DNA sequences to arrays.
In a fifth aspect, this invention features a method for the combinatorial synthesis of biopolymers at the test site microlocations or macrolocations
In an sixth aspect, this invention features a device which electronically delivers reagents and reactants with minimal use of fluidics.
In a seventh aspect, this invention features a device which carries out molecular biology and DNA amplification reactions (e.g. Polymerase Chain Reaction, Strand Displacement Amplification, restriction cleavage reactions, DNA/RNA polymerase and DNA ligase target amplification reactions.
In an eighth aspect, this invention features a device which can electronically size and identify restriction fragments (e.g. carry out electronic restriction fragment length polymorphism, short tandem repeat polymorphism and DNA finger printing analysis).
In an ninth aspect, this invention features a device which carries out antibody/antigen, immunodiagnostic reactions, and proteomic analysis.
In a tenth aspect, this invention features a device which is able to carry out combinatorial synthesis of oligonucleotides and peptides.
In an eleventh aspect, this invention features a device which selectively binds cells, processes cells for hybridization, causes cell lysis (electronic, hypotonic or hypertonic), removes DNA from cells, or carries out electronic in-situ hybridizations within the cells.
In a twelfth aspect, this invention features devices and methods which allow rapid multiplex hybridization and discrimination of single base mismatches (single nucleotide polymorphisms) in full length double-stranded or single-stranded DNA fragments, RNA fragments, PCR amplicons, and SDA amplicons, under what would normally be considered substantially non-hybridizing or denaturing conditions by any passive or conventional hybridization technique.
In a thirteenth aspect, this invention features electronic hybridization methods which incorporate buffer and/or electrolyte entity including but not limited to: histidine, di-histidine, histidine peptides, mixed histidine peptides, Na+, K+, Mg++, NH4+, amines and other double strand DNA stabilizing entities; which can allow rapid transport and hybridization of nucleic acid fragments (DNA, RNA, etc.) under what would normally be considered substantially non-hybridizing or denaturing conditions by any passive or conventional hybridization technique.
In a fourteenth aspect, this invention features devices and methods which allow rapid multiplex hybridization and discrimination of multiple repeat sequences (di-, tri, tetra, etc.), including short tandem repeats (STRs) in nucleic acid fragments, under what would normally be considered substantially non-hybridizing or denaturing conditions by any passive or conventional hybridization technique.
In a fifteenth aspect, this invention features devices and methods which allow rapid multiplex hybridization in in-situ formats.
In a sixteenth aspect, this invention features devices and methods which can be combined into an instrument system which allows use of various specific binding entities or reactants which would not be compatible under normal passive or conventional reaction conditions, including common temperature and buffer composition.
In a seventeenth aspect, this invention features devices and methods which allow rapid multiplex catalytic reactions.
In a eighteenth aspect, this invention features devices and methods which allow rapid multiplex organic synthesis reactions.
In a nineteenth aspect, this invention features devices and methods which allow rapid multiplex polymer synthesis.
In a twentieth aspect, this invention features devices and methods which allow rapid combinatorial synthesis and selection reactions.
In a twenty-first aspect, this invention features devices and methods which allow rapid multiplex nanofabrication reactions.
In a twenty-second aspect, this invention features devices and methods which integrate biosensors or electronic detection components within the test site microlocations.
In a twenty-third aspect, this invention features devices with alternative second entity motive forces, such as the use of high pressure forces rather than or in addition to electromotive forces to transport the second entity through the semi-permeable matrix.
The active, electronic nature of the devices of the invention allows us to create new mechanisms for carrying out a wide variety of molecular biological reactions. These include novel methods for achieving both linear and exponential multiplication or amplification of target DNA and RNA molecules.
The device provides electronic mechanisms to: (1) transport denatured DNA or other charged entities in bulk solution at room temperature (e.g. well below their Tm points); (2) to selectively concentrate the specific charged entities, (e.g., DNA targets or probes, reactants, reagents, and enzymes, etc.) at the desired test site microlocation; and (3) to subsequently create xe2x80x9creaction zonesxe2x80x9d of favorable environment and reaction conditions to greatly decrease the time necessary for hybridization and/or other common molecular biological reactions. These all involve new physical parameters for carrying out molecular biological and target amplification type reactions.
A number of examples of electronically controlled molecular biology reactions (can be) have been developed, these include: (1) Electronically Directed Restriction Enzyme Cleavage of Specific ds-DNA Sequences; (2) Electronic Restriction Fragment Analysis; (3) Electronic Multiplication of Target DNA by DNA Polymerases; and (4) Electronic Ligation and Multiplication of Target DNA Sequences By DNA and RNA Polymerases; and (5) Electronic Multiplication of Target DNA by RNA Polymerases. These examples are representative of the types of molecular biological reactions and procedures which can be carried but on the devices of the invention.
Other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.